Abstract The Specific Aim of this Phase I STTR proposal is to test the feasibility of treating non-alcohol steatohepatitis (NASH) by increasing mitochondrial metabolism, which is the main pathway for lipid catabolism in the liver. NASH is a stage of non-alcohol fatty liver disease (NAFLD) that has progressed to a pathological state. NASH can lead to cirrhosis, leading to liver failure or hepatocellular carcinoma. NASH is especially common in those with other metabolic disorders, such as obesity, diabetes, and/or hyperlipidemia, but also affects the healthy population. About 30% of the population in US has NAFLD, with 5 million instances of NASH diagnosed each year in the US with advanced fibrosis and about 1.8 million individuals progressed to cirrhosis. The primary treatment for NASH is lifestyle change. However, few patients remain adherent for extended periods of time. No drugs have been approved by the FDA for treating NASH. To address this need, we are developing an siRNA-based drug to treat NASH by increasing liver metabolism. Fatty acids are transported to the liver where they are metabolized through beta-oxidation coupled to the electron transport chain (ETC) in the mitochondria. Increased mitochondrial respiratory activity in the liver could therefore speed up the degradation of fatty acids and prevent their accumulation in the liver. A key regulator of the ECT is the MCJ protein (MCJ/DnaJC15 or Methylation-Controlled J protein). MCJ is a mitochondrial protein that reduces respiration dependent ATP levels. We have demonstrated that removal of MCJ activity is safe and results in increased mitochondrial metabolism/ATP production. We have developed an siRNA formulation (siMCJ) that effectively removes mouse MCJ in the liver. Using this formulation, we have shown in two mouse models that we can reverse or prevent NASH pathologies. siRNA has been validated as a drug for number of diseases, including liver diseases, with over 30 clinical trials having been run, thus validating the use of this therapeutic approach. While our preliminary data are compelling thus far, the studies were carried out at only one dose of siRNA and against the mouse MCJ protein. Thus, to show feasibility of our approach, prior to carrying out IND enabling studies, we must carry out a more robust feasibility study that includes demonstration of safety, a dose/effect relationship, and efficacy of human siMCJ. To this end, we will carry out the following Tasks: Task 1: Evaluate efficacy and toxicity of mouse siMCJ in vivo using mouse models Task 2: Evaluate efficacy human siMCJ in vivo using humanized-liver mouse model (KMT-Mice) Test of Feasibility: We must observe findings in Task 1 that are similar to those in the preliminary study section and we must observe a dose/effect curve that can be fit to a standard Hill type or log/logit analysis and that covers the EC50 point of the curve. We must observe a TI (therapeutic index) of at least 3 (maximally effective dose/MTD). This TI would indicate a very safe dosing window, since we use a maximally effective dose as the numerator. Findings in Task 2 must also be similar to the findings in the preliminary study section.